Periodic Acid-Schiff Reaction ---Histochemistry for glycogen (Aug 16, 2000)

 

·         PAS reaction was described by McManus in 1946

 

·         Mechanism---Two steps

1.       Oxidation with periodic acid to form aldehyde groups. (A short blockade in Dimedone, an aldehyde blocking agent, abolishes the PAS reaction of the vast majority of widely distributed materials such as connective tissue mucopolysaccharides and glycoproteins, while a much longer blockade is required to abolish the PAS reaction of glycogen)

2.       Demonstration of the aldehyde groups with Schiff’s reagent

 

·         Procedures

1.       Animals: Adult male SD rats (250-300g)

2.       Perfusion:

·         Cold 0.9% NaCl 30ml

·         Cold 4% paraformaldehyde in PB, pH 7.3 100ml

3.       Dissect out rat brains

4.       Post-fixation: Place rat brains in 4% paraformaldehyde overnight

5.       Transfer rat brain to 0.5M sucrose in PB, pH 7.3, overnight or longer, 4C

6.       Frozen 8 mm sections cut on a crystat and mounted on silane-treated slides

7.       Air-dry sections

8.       Arrange sections in a rack and bring to water, 1 min

·         For control, treat sections with malt diastase (Amylase) for 3 hour at 37^oC, then rinse in water.

·         Digestion solution:

-          Malt diastase                                    500 mg

-          0.02M Phosphate buffer, pH 6.0       50 ml

-          Sodium chloride                              400 mg

9.       0.5% periodic acid 10 min at RT.

·         0.5% periodic acid is reasonably stable, good for at least one month

10.   Wash in tap water, 3 min

11.   Treat sections in saturated solution of dimedone in distilled water, 20 min at 60^oC

12.   Wash in distilled water 5 min

13.   Treat with Schiff’s reagent, 15 min

·         Preparation of Schiff’s reagent:

-          Basic fuchsin 1 g in 200 ml water, boil to 100^oC for 5 in.

-          Cool down to 50^oC, filter it

-          Add 20 ml N-hydrochloric acid

-          Cool to 25^oC, add 1 g sodium metabisulphite

-          Store in dark overnight

-          Add 2 g activated charcoal, shake well for 1 min

-          Filter and store in dark at 4^oC

14.   Wash in distilled water, 1 min

15.   Wash in tap water, 3 min

16.   Dehydrate in graded alcohol (70%, 90%, 95%, 100%, 100%) to xylene

17.   Mount in DPX or Permount

 

·         Results:

  1.        PAS positive: Magenta

  2.        Diastase control: Negative

·         Note: Wash in distilled water before and after Schiff’s reagent is important

·         References

1.       Bulmer, D. (1959) Dimedone as an aldehyde blocking reagent to facilitate the histochemical demonstration of glycogen. Stain Technol 34:95-98